Micro-arrays are tools for protein, DNA and RNA molecular diagnostics, often used for the detection of nucleic acids and proteins. Such parallellisation techniques, enable the investigation of several thousands of sequences in one reaction or experiment. Most applications are focused on expression profiling for measuring adequately the expression of several thousands genes of interest. Presently, detection of molecular binding on micro-arrays is effected by use of special fluorescent dyes such as Cy3 and Cy5. However, the accuracy and reproducibility of such techniques is limited by the stability of the fluorescent marker.
Sequence-selective DNA detection has become increasingly important as scientists unravel the genetic basis of disease and use this new information to improve medical diagnosis and treatment. DNA hybridization tests on oligonucleotide-modified substrates are commonly used to detect the presence of specific DNA sequences in solution. The developing promise of combinatorial DNA arrays for probing genetic information illustrates the importance of these heterogeneous sequence assays to future science. In most assays, the hybridization of fluorophore-labeled targets to surface bound probes is monitored by fluoresecence microscopy or densitometry. Although fluorescence detection is very sensitive, its use is limited by the expense of the experimental equipment and by background emissions from most common substrates. In addition, the selectivity of labeled oligonucleotide targets for perfectly complementary probes over those with single base mismatches is poor, preventing the use of surface hybridization tests for detection of single nucleotide polymorphisms.
Another disadvantage is that the evaluation of processed fluorescence based micro-arrays needs highly expensive laser scanning devices, as well as highly sophisticated software for analysing the data generated by the laser scanning devices. Such equipment is not available to all laboratories, and certainly not to laboratories with limited resources. Thus a major drawback of this technology the capital expense, and its use restricted to well resourced laboratories.
A limited number of prior art documents describe detection methods and/or reagents for use in solid-phase assays, but each is associated with numerous disadvantages. Enzymatic methods or gold-based detection method on micro-arrays are described in WO 00/72018, EP 1164201, EP 1096024, WO 01/77372A2, US 2001/0010906A1. The patent application number WO 00/72018 describes the use of biotinylated DNA on micro-array using streptavidin labelled gold. Other patent documents such as EP 1164201, EP 1096024 and WO 01/77372A2 describe methods and kits for detection and quantification of homologue nucleotides, method and kit for diagnosis and quantification using sandwich hybridisation on solid carrier (US 2001/0010906A1). Patent document EP 1164201 describes the use of inverted detection for identifying and/or quantifying nucleotide target sequences on biochips using micro-fluidity techniques. Patent document EP 10960245 describes a method for detection of homologue sequences after multiplex PCR for detecting Staphylococcus microorganisms. Patent documents AU8366001, AU7547501, CA2397280, WO 01/96604 and WO 99/20789 describe a detection technique using streptavidin labelled gold particles of at least 80 nm for visualisation of bound nucleic acids on micro-array. Patent document U.S. Pat. No. 6,451,980 describes a technique for signal enhancement using a bi-specific antibody-polymer probe, wherein one part of said probe recognises the antigen and the other part is directed against DTPA (diethylenepentaacteic acid). Patent document WO 02/06511 (Genisphere) describes an amplification technique applicable to micro-arrays using a DNA dendrimer-based approach. Patent document WO 01/36681 (Digene) discloses a technology for detecting DNA/RNA hybrids on micro-arrays using a monoclonal antibody directed specifically to RNA/DNA hybrids with visualisation using fluorescent dyes. Patent document WO 96/14314 (DAKO) describes the use of a specific monoclonal antibody for detecting DNA/PNA nucleic acid hybrids. The use of lectins or dextran coated with biotin molecules for use specially in in situ hybridization is disclosed in patent document EP0151492 (ENZO).
A detection scheme which improves upon the simplicity, sensitivity and selectivity of methods in the art could allow the full potential of combinatorial sequence analysis to be realized. Therefore, for reading and analysing arrays at a low cost, with high sensitivity and reproducibility, there is a need for a technique which overcomes the problems of the art.
For generalizing the use of this technology, it would be advantageous to have a visualization technique that enables the investigator to evaluate the hybridization results with the naked eye without use of laser scanning devices or other kind of expensive visualization equipment. Alternatively, a system which enables the use of low cost readers e.g. those which detect electrical conductance and/or changes in magnetic flux would help sensitive array-type assaying become more cost effective as a research and diagnostic tool.